Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is an enzyme, which catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules. Glucoamylases are produced by several filamentous fungi and yeasts, with those from Aspergillus being commercially most important.
Commercially, the glucoamylase enzyme is used to convert corn starch which is already partially hydrolyzed by an alpha-amylase to glucose. The glucose is further converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world's largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced.
One of the main problems that exist with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylase. Glucoamylase is not as thermally stable as alpha-amylase or glucose isomerase and it is most active and stable at lower pH's than either alpha-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.
Glucoamylase from Aspergillus niger has a catalytic (aa 1-440) and a starch binding domain (aa 509-616) separated by a long and highly O-glycosylated linker (Svensson et al. (1983), Carlsberg Res. Commun. 48, 529-544, 1983 and (1986), Eur. J. Biochem. 154, 497-502). The catalytic domain (aa 1-471) of glucoamylase from A. awamori var. X100 adopt an (alpha/alpha)6-fold in which six conserved alpha→alpha loop segments connect the outer and inner barrels (Aleshin et al. (1992), J. Biol. Chem. 267, 19291-19298). Crystal structures of glucoamylase in complex with 1-deoxynojirimycin (Harris et al. (1993), Biochemistry, 32, 1618-1626) and the pseudotetrasaccharide inhibitors acarbose and D-gluco-dihydroacarbose (Aleshin et al. (1996), Biochemistry 35, 8319-8328) furthermore are compatible with glutamic acids 179 and 400 acting as general acid and base, respectively. The crucial role of these residues during catalysis have also been studied using protein engineering (Sierks et al. (1990), Protein Engng. 3, 193-198; Frandsen et al. (1994), Biochemistry, 33, 13808-13816). Glucoamylase-carbohydrate interactions at four glycosyl residue binding subsites, −1, +1, +2, and +3 are highlighted in glucoamylase-complex structures (Aleshin et al. (1996), Biochemistry 35, 8319-8328) and residues important for binding and catalysis have been extensively investigated using site-directed mutants coupled with kinetic analysis (Sierks et al. (1989), Protein Engng. 2, 621-625; Sierks et al. (1990), Protein Engng. 3, 193-198; Berland et al. (1995), Biochemistry, 34, 10153-10161; Frandsen et al. (1995), Biochemistry, 34, 10162-10169.
Different substitutions in A. niger glucoamylase to enhance the thermal stability have been described: i) substitution of alpha-helical glycines: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505); ii) elimination of the fragile Asp-X peptide bonds, D257E and D293E/Q (Chen et al. (1995), Prot. Engng. 8, 575-582); prevention of deamidation in N182 (Chen et al. (1994), Biochem. J. 301, 275-281); iv) engineering of additional disulphide bond, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and v) introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204. Furthermore Clark Ford presented a paper on Oct. 17, 1997, ENZYME ENGINEERING 14, Beijing/China Oct. 12-17, 1997, Abstract number: Abstract book p. 0-61. The abstract suggests mutations in positions G137A, N20C/A27C, and S30P in an (not disclosed) Aspergillus awamori glucoamylase to improve the thermal stability.